Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

II. Neutrophil Functions
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the v- and t-SNAREs. When separated, SNAREs from
apposed membranes are paired resulting in fusion of the
lipid bilayers. However, fusion occurs in a highly controlled
manner being regulated primarily by an Src family
of RAB GTPases ( Peyron et al. , 2001 ).
Phagolysosome fusion is a critical event in killing of
pathogenic organisms. Defective phagolysosome fusion
occurs in Chediak-Higashi syndrome. Chediak-Higashi syndrome
has been described in several breeds of cattle, Persian
cats, foxes, mink, and a killer whale ( Ayers et al. , 1988 ;
Colgan et al. , 1992 ). Humans and animals have partial oculocutaneous
albinism, frequent infections, and mild bleeding
diatheses. Neutrophils, monocytes, and melanocytes contain
giant granules that result of abnormal fusion events that
appear to be due to defects in lysosomal docking proteins.
Granule membrane fusion is defective and discharge of
granule contents into the phagosome is delayed ( Mills and
Noya, 1993 ).
C . Bactericidal Mechanisms
The mature phagolysosome has multiple mechanisms to kill
microorganisms. These have generally been divided into
oxygen-dependent and oxygen-independent mechanisms
( Klebanoff, 1975 ).
1 . Oxygen-Dependent Mechanisms
Oxygen-dependent mechanisms are located within primary
granules ( Babior, 1984 ; Babior et al. , 2002 ). These
mechanisms are initiated by the process of phagocytosis or
by perturbation of the cell membrane. A membrane-bound
nicotinamide-adenine dinucleotide phosphate (NADPH)
oxidase consists of five essential protein components; two of
these are in the membrane and three are in the cytosol ( Fig.
11-4 ). The membrane-associated component is a flavocytochrome
b 558 that consists of a heterodimer composed of 91
(gp91 phox ) and 22 (gp22 phox ) kDa proteins. Flavocytochrome
b 558 is distributed in the cell membrane and in the membrane
of the granules and is incorporated into the wall of
phagocytic vacuoles. The cytosolic components consist of a
40-kDa protein (p40- phox ), a 47-kDa protein (p47- phox )
and a 67-kDa component (p67- phox ). When stimulated, the
cytosolic components are translocated to the membrane.
Both the assembly of the NADPH oxidase complex and the
electron flow are dependent on the influence of three GTPbinding
proteins; Rac1, Rac2, and Rap1A. The NADPH oxidase
complex catalyzes the reduction of molecular oxygen to
superoxide anion (NADPH O 2 →NADP H O 2
).
The associated rapid consumption of oxygen has been
termed the “ respiratory burst. ” Superoxide anions are rapidly
dismutated to hydrogen peroxide (H 2 O 2 ). Superoxide anions
also can be converted to hydroxyl radicals (OH . ) in the presence
of catalytic metals such as iron according to the Haber-
Weiss reaction (Fe 2 H 2 O 2 →Fe 3 OH OH . O 2 ).
O 2
p22
rac
p47
p67
p40
gp 91
phox
NADPH
O 2
NADP H
FIGURE 11-4 The NADPH oxidase complex consists of a membrane-bound
flavocytochrome b 558 consisting of two subunits (22 and
91kDa) and three cytosolic molecules (p40, p47, and p67). The assembly
of NADPH and electron flow are dependent on three GTP-binding
proteins (Rac).
Myeloperoxidase catalyzes the conversion of H 2 O 2 and
halide ions to hypohalous acids with hypochlorous acid
being the primary acid produced. Finally H 2 O 2 interacts
with hypohalous acids to produce singlet oxygen ( 1 O 2 ).
Singlet oxygen is a high-energy form of oxygen that can
attack double bonds. These reactive oxygen intermediates
interact with unsaturated lipids, carbon bonds, sulfhydryl
and amino groups, nucleic acids, pyrimidine nucleotides,
and enzymes within organisms. The extent to which these
events contribute directly to disruption of organism function
and organism killing is controversial ( Segal, 2005 ;
this subject is discussed in more detail in the next section).
Myeloperoxidase is not essential for antimicrobial activity
because humans with myeloperoxidase deficiency do not
have severe bacterial infections. Additionally chicken heterophils
lack myeloperoxidase.
Products of the respiratory burst are toxic to cells
as well as to invading organisms. A variety of antioxidant
systems exist to protect cells from the harmful effect of
these oxidants ( VanSteenhouse, 1987 ). These systems
include cytosolic superoxide dismutase, glutathione peroxidase,
glutathione reductase, and catalase, all of which convert
oxygen radicals to water. Other compounds with oxygen
radical scavenging potential including vitamin E, vitamin C,
selenium, transferrin, and cysteine ( VanSteenhouse, 1987 ).
The glutathione needed for the reaction is maintained in the
reduced state by reduced NADPH that is supplied by the
hexose monophosphate pathway.
Several defects in the capacity of neutrophils to generate
oxygen radicals have been described. These include
chronic granulomatous disease, glutathione peroxidase
deficiency, glucose-6-phosphatase deficiency, and myeloperoxidase
deficiency ( Malech and Nauseef, 1997 ).
Chronic granulomatous disease is caused by mutations
in genes encoding components of NADPH oxidase. In
excess of 40 specific mutations have been reported in human
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